Dye-based probes for protein activation in living cells

描述（由申请人提供）：将开发新的方法来检查活细胞中信号蛋白激活的动力学。这些方法将基于新的、环境敏感的荧光染料，其设计用于响应体内蛋白质构象变化。一般适用的方法和染料将通过设计Cdc42，Rac和Rho的核苷酸状态的生物传感器来开发。新方法将提供研究活细胞中内源性未标记蛋白质的能力，研究同一细胞中多种蛋白质的激活，并检查埋藏在多蛋白复合物中的蛋白质。染料将非常明亮，并经历大的荧光变化，使我们能够使用低浓度的生物传感器，以最小限度地干扰正常细胞生理学。这些染料可用于在光漂白之前获得许多图像，提供优异的时间分辨率，并定量单个细胞中蛋白质活化水平随时间的变化。Rac和Cdc42的生物传感器将在同一细胞中成像，以检查Rac的Cdc42激活如何协调两种GTP酶的活性以实现运动性。最后，我们将开发将抗体片段转化为生物传感器的方法，从而提供许多蛋白质活性。新的染料将使使用高度可逆的抗体结合成为可能，以最小限度地干扰细胞行为。对磷酸化α 4整联蛋白特异性的单克隆抗体将用作开发该方法的模型，并研究α 4对Rac在定向细胞运动中的调节。

DESCRIPTION (provided by applicant): New approaches will be developed to examine the dynamics of signaling protein activation in living cells. These methods will be based upon novel, environmentally-sensitive fluorescent dyes designed to respond to protein conformational changes in vivo. Generally applicable methods and dyes will be developed by designing biosensors for the nucleotide state of Cdc42, Rac, and Rho. The new approaches will provide the ability to study endogenous, unlabeled proteins in living cells, to study activation of multiple proteins in the same cell, and to examine proteins buried within multiprotein complexes. The dyes will be very bright and undergo large fluorescence changes, enabling us to use low biosensor concentrations for minimum perturbation of normal cell physiology. The dyes can be used to obtain many images before photobleaching, providing excellent temporal resolution, and quantitation of changes in protein activation level over time in individual cells. Biosensors of Rac and Cdc42 will be imaged in the same cell, to examine how Cdc42 activation of Rac coordinates the activity of the two GTPases for motility. Finally, we will develop means to convert antibody fragments into biosensors, providing access to many protein activities. The new dyes will make it possible to use highly reversible antibody binding, to minimally perturb cell behavior. A monoclonal antibody specific for phosphorylated alpha4 integrin will be used as a model for development of the approach, and to study alpha4 regulation of Rac in directed cell movement.